Novel human integral membrane protein

ABSTRACT

The present invention provides a novel human integral membrane (IMP-2) and polynucleotides which identify and encode IMP-2. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding IMP-2 and a method for producing IMP-2. The invention also provides for agonists, antibodies, or antagonists specifically binding IMP-2, and their use, in the prevention and treatment of diseases associated with expression of IMP-2. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding IMP-2 for the treatment of diseases associated with the expression of IMP-2. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding IMP-2.

[0001] This application is a continuation application of U.S.application Ser. No. 09/207,161, filed Dec. 7, 1998, entitled NOVELHUMAN INTEGRAL MEMBRANE PROTEIN, which is a divisional application ofU.S. application Ser. No. 08/791,338, filed Jan. 31, 1997, now U.S. Pat.No. 5,889,170, issued Mar. 30, 1999, entitled HUMAN INTEGRAL MEMBRANEPROTEIN, the contents all of which are hereby incorporated herein byreference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a novel integral membrane protein, IMP-2, and to the use of thesesequences in the diagnosis, prevention, and treatment of disease.

BACKGROUND OF THE INVENTION

[0003] Membrane proteins are divided into two groups based upon the easewith which the proteins can be removed from the membrane. Extrinsic orperipheral membrane proteins can be removed using extremes of ionicstrength or pH, the use of urea or other disruptors of proteininteractions. Intrinsic or integral membrane proteins are released onlywhen the lipid bilayer of the membrane is dissolved by detergent.Extrinsic membrane proteins comprise the constituents of thecytoskeleton such as spectrin and actin. Many cytoskeletal proteins arebound directly to integral membrane proteins or are bound indirectly viaother proteins such as ankyrin. Cytoskeletal proteins control the shapeand dynamics of the cell membrane through their interactions with motorproteins such as myosin and dynein.

[0004] The majority of known integral membrane proteins aretransmembrane proteins which comprise an extracellular, a transmembrane,and an intracellular domain. Transmembrane proteins are typicallyembedded into the cell membrane by one or more regions comprising 15 to25 hydrophobic amino acids which are predicted to adopt an α-helicalconformation. Transmembrane proteins are classified as bitopic (or TypesI and II) and polytopic (or Types III and IV) [Singer, S. J. (1990)Annu. Rev. Cell Biol. 6:247-96]. Bitopic proteins span the membrane oncewhile polytopic proteins contain multiple membrane-spanning segments. Asmall number of integral membrane proteins, termed monotopic proteins,are partially embedded in the membrane (i.e., they do not span the lipidbilayer). Monotopic proteins may be inserted into the bilayer by ahydrophobic hairpin loop or may be attached to the membrane via boundlipid.

[0005] Type II integral membrane proteins have a single transmembranestretch of hydrophobic residues which is often located near theamino-terminus. The bulk of type II proteins comprises thecarboxy-terminal domain which is located on the exterior side of thecell. The amino-terminal domain of type II proteins, located on thecytoplasmic side of the cell membrane, is typically small. Thus, thetype II proteins generally lack enzymatically active domains on thecytoplasmic side of the membrane and thus are not themselves directlyinvolved in transmembrane signalling. The carboxy-terminus of type IIproteins typically comprises the active portion of the protein (e.g.,the active site of an enzyme, the binding domain of a receptor).

[0006] Recently a multigene family encoding type II integral membraneproteins, termed the E25 gene family, was identified [Deleersnijder W etal. (1996) J. Biol. Chem. 271:19475]. The best characterized member ofthis family is the mouse Itm2 gene which encodes the E25AMM protein. Theexpression of the Itm2 gene was found to be associated withchondro-osteogenic differentiation. The Itm2 gene is strongly, althoughnot exclusively, expressed in osteogenic tissue. In particular, Itm2 isstrongly expressed in mature osteoblasts and in early stages ofsecondary chondrogenesis. Itm2 expression is not limited tochondro-osteogenic tissues as it is expressed in 1) heart, brain(choroid plexus), renal cortex, and the crypts of the small intestine(weak expression) and in 2) skin (stratum corneum), hair follicles andthe acini of exocrine glands (strong expression) [Deleersnijder W etal., supra]. Additional members of the E25 multigene family have beenidentified in the mouse and in humans. These additional E25 familymembers are expressed in a wide variety of tissues including adult andfetal brain, fetal liver, fetal spleen, lung, breast, placenta,prostate, adrenal gland, white blood cells and adult and fetal heart[Deleersnijder W et al., supra].

[0007] The discovery of molecules related to the E25 multigene familysatisfies a need in the art by providing new diagnostic or therapeuticcompositions useful in the treatment of disorders associated withalterations in the expression of members of the E25 multigene family.

SUMMARY OF THE INVENTION

[0008] The present invention features a novel integral membrane proteinhereinafter designated IMP-2 and characterized as having similarity tothe mouse E25AMM integral membrane protein.

[0009] Accordingly, the invention features a substantially purifiedpolypeptide having the amino acid sequence shown in SEQ ID NO: 1 orfragments thereof. Preferred fragments of SEQ ID NO: 1 are fragments ofabout 15 amino acids or greater in length which define fragments unique(i.e., having less than about 25% identity to fragments of anotherprotein) to SEQ ID NO: 1 or which retain biological activity orimmunological activity (i.e., capable of eliciting anti-IMP-2antibodies). Fragments of SEQ ID NO: 1 which are at least 25 aminoacids, at least 50 amino acids, at least 100 amino acids, at least 125amino acids and at least 200 amino acids in length are contemplated.

[0010] The present invention further provides isolated and substantiallypurified polynucleotide sequences encoding the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 or fragments thereof. In aparticular aspect, the polynucleotide is the nucleotide sequence of SEQID NO: 2 or variants thereof. In another embodiment, the presentinvention provides polynucleotides comprising fragments of SEQ ID NO: 2having a length greater than 20 nucleotides. The invention furthercontemplates fragments of this polynucleotide sequence (i.e., SEQ ID NO:2) that are at least 50 nucleotides, at least 100 nucleotides, at least250 nucleotides, at least 500 nucleotides and at least 1000 nucleotidesin length.

[0011] In addition, the invention provides polynucleotide sequenceswhich hybridize under stringent conditions to the polynucleotidesequence of SEQ ID NO: 2. In another embodiment the present inventionprovides a composition comprising an isolated and purifiedpolynucleotide sequence encoding IMP-2.

[0012] The invention provides polynucleotide sequences comprising thecomplement of SEQ ID NO: 2 or variants thereof; these complementarynucleic acid sequences may comprise the complement of the entire nucleicacid sequence of SEQ ID NO: 2 or fragments thereof. In anotherembodiment the present invention provides a composition comprising anisolated and purified polynucleotide sequence comprising the complementof SEQ ID NO: 2 or variants thereof.

[0013] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode IMP-2.

[0014] In another embodiment the present invention provides an isolatedpolynucleotide comprising at least a portion of the nucleic acidsequence of SEQ ID NO: 2 or variants thereof contained on a recombinantexpression vector. In yet another embodiment, the expression vectorcontaining the polynucleotide sequence is contained within a host cell.The invention is not limited by the nature of the host cell employed.For example, the host cell may be an E. coli cell, a yeast cell, aninsect cell, a mammalian cell, etc.

[0015] The present invention also provides a method for producing apolypeptide comprising the amino acid sequence of SEQ ID NO: 1 orfragments thereof, the method comprising the steps of: a) culturing thehost cell containing an expression vector containing an isolatedpolynucleotide encoding at least a fragment of the IMP-2 polypeptideunder conditions suitable for the expression of the polypeptide; and b)recovering the polypeptide from the host cell culture.

[0016] In another embodiment, the invention provides a pharmaceuticalcomposition comprising a substantially purified human IMP-2 proteinhaving the amino acid sequence of SEQ ID NO: 1 in conjunction with asuitable pharmaceutical carrier.

[0017] The invention also provides a purified antibody which bindsspecifically to a polypeptide comprising at least a portion of the aminoacid sequence of SEQ ID NO: 1.

[0018] Still further, the invention provides a purified agonist whichspecifically binds to and modulates the activity of a polypeptidecomprising at least a portion of the amino acid sequence of SEQ IDNO: 1. The present invention further provides a pharmaceuticalcomposition comprising a purified agonist which specifically binds toand modulates the activity of a polypeptide comprising at least aportion of the amino acid sequence of SEQ ID NO: 1. In anotherembodiment, the invention provides a purified antagonist whichspecifically binds to and modulates the activity of a polypeptidecomprising at least a portion of the amino acid sequence of SEQ IDNO: 1. The present invention further provides a pharmaceuticalcomposition comprising a purified antagonist which specifically binds toand modulates the activity of a polypeptide comprising at least aportion of the amino acid sequence of SEQ ID NO: 1.

[0019] The invention also provides a method for treating liver disease(including liver tumors) comprising administering to a subject in needof such treatment an effective amount of a pharmaceutical compositioncomprising a purified agonist which specifically binds to and modulatesthe activity of a polypeptide comprising at least a portion of the aminoacid sequence of SEQ ID NO: 1. The treatment of a variety of tumors,including but not limited to tumors of the lung, prostate, breast andbladder, using agonists as well as antagonists of IMP-2 is alsocontemplated by the present invention.

[0020] The invention also provides a method for detection ofpolynucleotides encoding human IMP-2 in a biological sample comprisingthe steps of: a) hybridizing a polynucleotide sequence encoding humanIMP-2 (SEQ ID NO: 1) to nucleic acid material of a biological sample,thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding human IMP-2 in thebiological sample. In a preferred embodiment, prior to hybridization,the nucleic acid material of the biological sample is amplified by thepolymerase chain reaction.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO: 1) andnucleic acid sequence (SEQ ID NO: 2) of IMP-2. The alignment wasproduced using MacDNASIS PRO™ software (Hitachi Software EngineeringCo., Ltd., San Bruno, Calif.).

[0022]FIG. 2 shows the amino acid sequence alignments among IMP-2 (SEQID NO: 1) and E25AMM (GI 624778; SEQ ID NO: 3). The alignment wasproduced using the multisequence alignment program of DNASTAR™ software(DNASTAR Inc, Madison Wis.).

[0023]FIGS. 3A and 3B show the hydrophobicity plots (MACDNASIS PROsoftware) for IMP-2, (SEQ ID NO: 1) and E25AMM (SEQ ID NO: 3); thepositive X axis reflects amino acid position, and the negative Y axis,hydrophobicity.

[0024]FIGS. 4A, 4B, 4C, and 4D show the northern analysis for SEQ ID NO:2. The northern analysis was produced electronically using LIFESEQ™database (Incyte Genomics, Inc., Palo Alto, Calif.).

DESCRIPTION OF THE INVENTION

[0025] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0026] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0027] Unless defined otherwise, all technical and scientific terms usedherein, have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein, can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

[0028] “Nucleic acid sequence” as used herein, refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein, refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0029] A “composition comprising a given polynucleotide sequence” asused herein, refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encodingIMP-2 (SEQ ID NO: 1) or fragments thereof (e.g., SEQ ID NO: 2 andfragments thereof) may be employed as hybridization probes. In thiscase, the IMP-2-encoding polynucleotide sequences are typically employedin an aqueous solution containing salts (e.g., NaCl), detergents (e.g.,SDS) and other components (e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.).

[0030] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0031] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0032] IMP-2, as used herein, refers to the amino acid sequences ofsubstantially purified IMP-2 obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0033] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCRTM (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

[0034] A “variant” of IMP-2, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0035] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0036] An “insertion” or “addition”, as used herein, refer to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0037] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0038] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic IMP-2, orany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0039] The term “agonist”, as used herein, refers to a molecule which,when bound to IMP-2, causes a change in IMP-2 which modulates theactivity of IMP-2. Agonists may include proteins, nucleic acids,carbohydrates, or any other molecules which bind to IMP-2.

[0040] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to IMP-2, blocks or modulates the biologicalor immunological activity of IMP-2. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to IMP-2.

[0041] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of IMP-2. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties of IMP-2.

[0042] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of IMP-2or portions thereof and, as such, is able to effect some or all of theactions of IMP-2-like molecules.

[0043] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding IMP-2 or the encoded IMP-2.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0044] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0045] “Amplification” as used herein, refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0046] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0047] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., Cot or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0048] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

[0049] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0050] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0051] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences. Under “stringentconditions” SEQ ID NO: 2 or fragments thereof will hybridize to itsexact complement and closely related sequences. The stringent conditionsare chosen such that SEQ ID NO: 2 or fragments thereof will hybridize tosequences encoding human IMP-2 but not to sequences encoding mouseE25AMM (i.e., SEQ ID NO: 4 or its RNA equivalents). When fragments ofSEQ ID NO: 2 are employed in hybridization reactions, the stringentconditions include the choice of fragments of SEQ ID NO: 2 to be used.Fragments of SEQ ID NO: 2 which contain unique sequences (i.e., regionswhich are either non-homologous to or which contain less than about 50%homology or complementarity with SEQ ID NO: 4) are preferentiallyemployed. SEQ ID NO: 4 represents DNA sequences encoding the mouseE25AMM protein; this DNA sequence can be found in GenBank underaccession number 624778.

[0052] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0053] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ ID NO:1” encompasses the full-length human IMP-2 and fragments thereof.“Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0054] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0055] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0056] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding IMP-2or fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0057] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO: 2 by northern analysis is indicativeof the presence of mRNA encoding IMP-2 in a sample and therebycorrelates with expression of the transcript from the polynucleotideencoding the protein. “Alterations” in the polynucleotide of SEQ ID NO:2, as used herein, comprise any alteration in the sequence ofpolynucleotides encoding IMP-2 including deletions, insertions, andpoint mutations that may be detected using hybridization assays.Included within this definition is the detection of alterations to thegenomic DNA sequence which encodes IMP-2 (e.g., by alterations in thepattern of restriction fragment length polymorphisms capable ofhybridizing to SEQ ID NO: 2), the inability of a selected fragment ofSEQ ID NO: 2 to hybridize to a sample of genomic DNA (e.g., usingallele-specific oligonucleotide probes), and improper or unexpectedhybridization, such as hybridization to a locus other than the normalchromosomal locus for the polynucleotide sequence encoding IMP-2 (e.g.,using fluorescent in situ hybridization [FISH] to metaphase chromosomespreads).

[0058] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind IMP-2polypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0059] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The Invention

[0060] The invention is based on the discovery of a novel human integralmembrane protein (IMP-2), the polynucleotides encoding IMP-2, and theuse of these compositions for the diagnosis, prevention, or treatment ofdiseases associated with abnormal liver tissue, including liver tumors.In addition, as mRNA encoding IMP-2 is found in a number of othertumors, IMP-2 serves as a marker for cancerous cells, particularlybrain, prostate, breast and bladder tumor cells.

[0061] Nucleic acids encoding the human IMP-2 of the present inventionwere first identified in Incyte Clone 632664 from the NEUTGMT01 cDNAlibrary through a computer-generated search for amino acid sequencealignments. A consensus sequence, SEQ ID NO: 2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 436536 (THYRNOT01), 632664 (NEUTGMT01), 1301662 (BRSTNOT07),2278468 (PROSNON01), and 2353669 (LUNGNOT20).

[0062] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1, as shown in FIGS. 1Aand 1B. IP-2 is 266 amino acids in length and contains nine cysteineresidues (i.e., C₃₈, C₅₄, C₅₆, C₅₇, C₈₉, C₁₆₄, C₂₂₃, C₂₄₈, and C₂₆₅). Inaddition to providing sites for disulfide bond formation, the cysteineresidues provide potential sites for palmitoylation. Five of the ninecysteine residues found in human IMP-2 are conserved in location withcysteine residues found in the mouse E25AMM protein (i.e., C₅₆, C₁₆₄,C₂₂₃, C₂₄₈, and C₂₆₅ of IMP-2). The human IMP-2 of the present inventioncontains numerous potential O-linked glycosylation sites (i.e., serineand threonine residues). IMP-2 has a single potential N-linkedglycoslyation site (i.e., Asn-X-Ser/Thr) (i.e., N₁₇₀) which is conservedin location with the single N-linked glycoslyation site found in themouse E25AMM protein (Deleersnijder et al., supra). In addition, thehuman IMP-2 of the present invention contains numerous potentialphosphorylation sites (i.e., typically the hydroxyl groups of serine,threonine and tyrosine residues although asparagine, histidine andlysine residues may also be phosphorylated), including a potential sitefor phosphorylation by cAMP-dependent protein kinase (e.g., R-X-S/T)(i.e., T₂₃₆).

[0063] The IMP-2 protein of the present invention, like the mouse E25AMMprotein, has an acidic isoelectric point (pI) (IMP-2 has a pI of 4.86and E25AMM has a pI of 5.41). In addition, the IMP-2 protein of thepresent invention, like the mouse E25AMM protein, has a high content ofleucine and isoleucine residues (IMP-2 contains 9% leucine and 9%isoleucine; E25AMM contains 10.2% leucine and 8% isoleucine).

[0064] IMP-2 contains a stretch of hydrophobic amino acid residues atpositions 53-76 which presumably forms the membrane spanning domain. Asillustrated by FIGS. 3A and 3B and 4A, 4B, 4C and 4D, IMP-2 and E25AMMhave similar hydrophobicity plots.

[0065] IMP-2 has chemical and structural homology with the mouse E25AMMprotein (GI 624778; SEQ ID NO: 3) (Deleersnijder et al., supra). Inparticular, IMP-2 and E25AMM share 39.8% identity overall and 49%identity and 71% similarity over the carboxy-terminal domians (residues115-265 of IMP-2 and residues 111-261 of E25AMM). A pair of residues aresaid to be similar if they represent conservative substitutions. FIG. 2provides an alignment between the amino acid sequences of SEQ ID NOS: 1and 3.

[0066] Northern analysis shows the expression of IMP-2-encodingsequences in various libraries, at least 24% of which are cancerous orimmortalized and at least 17% of which are involved with the immuneresponse, including inflammatory and/or autoimmune disease (e.g.,rheumatoid synovium, ulcerative colitis, Crohn's disease, primarybiliary cirrhosis). Of particular note is the expression of IMP-2 mRNAin brain tumor (7/214), prostate tumor (6/214), breast tumor (3/214) andbladder tumor (3/214) libraries. This pattern of expression demonstratesthat IMP-2 serves as a marker for cancerous cells, particularly brainand prostate tumor cells. In addition to its expression in a variety oftumors, IMP-2 is highly expressed in adult liver and fetal spleen andthus serves as a marker for these tissues.

[0067] IMP-2 cDNA is strongly expressed in normal adult liver (>10%abundance; see LIVRNOT01 and LIVRNOM01 libraries) and its expressiondecreases precipitously in abnormal liver tissues, including primarybiliary cirrhosis (see LIVRBCT01 library; 3.5% abundance) and livertumors (see LIVRTUT01 library; 0.03% abundance). Thus, decreased or lowlevel (i.e., less than about 50% the level seen in normal ordisease-free, liver tissue) expression of IMP-2 in liver tissue servesas an indicator of liver disease, including liver cancer. A similardecrease in IMP-2 transcripts is observed when normal lung and lungtumors are compared; lung tumors show about a 50% decrease in IMP-2transcript abundance as compared to normal adult lung. Thus, decreasedor low level expression of IMP-2 in lung tissue serves as an indicatorof lung tumors.

[0068] The invention also encompasses IMP-2 variants. A preferred IMP-2variant is one having at least 80%, and more preferably 90%, amino acidsequence similarity to the IMP-2 amino acid sequence (SEQ ID NO: 1). Amost preferred IMP-2 variant is one having at least 95% amino acidsequence similarity to SEQ ID NO: 1.

[0069] The invention also encompasses polynucleotides which encodeIMP-2. Accordingly, any nucleic acid sequence which encodes the aminoacid sequence of IMP-2 can be used to generate recombinant moleculeswhich express IMP-2. In a particular embodiment, the inventionencompasses the polynucleotide comprising the nucleic acid sequence ofSEQ ID NO: 2 as shown in FIGS. 1A and 1B.

[0070] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding IMP-2, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring IMP-2, and all suchvariations are to be considered as being specifically disclosed.

[0071] Although nucleotide sequences which encode IMP-2 and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring IMP-2 under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding IMP-2 or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding IMP-2 and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0072] The invention also encompasses production of DNA sequences, orportions thereof, which encode IMP-2 and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding IMP- or any portionthereof.

[0073] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO: 2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0074] Altered nucleic acid sequences encoding IMP-2 which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent IMP-2. The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent IMP-2. Deliberate amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of IMP-2 is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine.

[0075] Also included within the scope of the present invention arealleles of the genes encoding IMP-2. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0076] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0077] The nucleic acid sequences encoding IMP-2 may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0078] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth, MN),or another appropriate program, to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0079] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-1 19). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0080] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0081] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0082] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0083] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode IMP-2, or fusion proteins orfunctional equivalents thereof, may be used in recombinant DNA moleculesto direct expression of IMP-2 in appropriate host cells. Due to theinherent degeneracy of the genetic code, other DNA sequences whichencode substantially the same or a functionally equivalent amino acidsequence may be produced and these sequences may be used to clone andexpress IMP-2.

[0084] As will be understood by those of skill in the art, it may beadvantageous to produce IMP-2-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0085] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterIMP-2 encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0086] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding IMP-2 may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of IMP-2 activity, it may be useful toencode a chimeric IMP-2 protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the IMP-2 encoding sequence and theheterologous protein sequence, so that IMP-2 may be cleaved and purifiedaway from the heterologous moiety.

[0087] In another embodiment, sequences encoding IMP-2 may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of IMP-2, or a portion thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 43 1A Peptide Synthesizer (Perkin Elmer).

[0088] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of IMP-2, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0089] In order to express a biologically active IMP-2, the nucleotidesequences encoding IMP-2 or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0090] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding IMP-2and appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0091] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding IMP-2. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0092] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector--enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding IMP-2,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0093] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for IMP-2. For example, whenlarge quantities of IMP-2 are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding IMP-2 may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0094] In the yeast, Saccharomvces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544. In caseswhere plant expression vectors are used, the expression of sequencesencoding IMP-2 may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S and 19S promoters of CaMV maybe used alone or in combination with the omega leader sequence from TMV(Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoterssuch as the small subunit of RUBISCO or heat shock promoters may be used(Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.Cell Differ. 17:85-105). These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.Such techniques are described in a number of generally available reviews(see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0095] An insect system may also be used to express IMP-2. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding IMP-2may be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of IMP-2 will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which IMP-2 may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0096] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding IMP-2 may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing IMP-2 in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0097] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding IMP-2. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding IMP-2, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0098] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO (ATCC CCL 61 and CRL 9618),HeLa (ATCC CCL 2), MDCK (ATCC CCL 34 and CRL 6253), HEK 293 (ATCC CRL1573), WI-38 (ATCC CCL 75) (ATCC: American Type Culture Collection,Rockville, Md.), which have specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

[0099] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress IMP-2 may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0100] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0101] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding IMP-2 isinserted within a marker gene sequence, recombinant cells containingsequences encoding IMP-2 can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding IMP-2 under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0102] Alternatively, host cells which contain the nucleic acid sequenceencoding IMP-2 and express IMP-2 may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0103] The presence of polynucleotide sequences encoding IMP-2 can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding IMP-2.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding IMP-2 todetect transformants containing DNA or RNA encoding IMP-2. As usedherein, “oligonucleotides” or “oligomers” refer to a nucleic acidsequence of at least about 10 nucleotides and as many as about 60nucleotides, preferably about 15 to 30 nucleotides, and more preferablyabout 20-25 nucleotides, which can be used as a probe or amplimer.

[0104] A variety of protocols for detecting and measuring the expressionof IMP-2, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson IMP-2 is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0105] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding IMP-2include oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding IMP-2, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used,include radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0106] Host cells transformed with nucleotide sequences encoding IMP-2may be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode IMP-2 may be designed to contain signal sequences which directsecretion of IMP-2 through a prokaryotic or eukaryotic cell membrane.When it is desired to express a secreted form of IMP-2, a polynucleotidesequence encoding a portion of the IMP-2 lacking the hydrophobic stretchlocated at residues 53-76 of SEQ ID NO: 1 (this stretch may anchor IMP-2in the membrane) is preferentially employed.

[0107] Other recombinant constructions may be used to join sequencesencoding IMP-2 to nucleotide sequences encoding a polypeptide domainwhich will facilitate purification of soluble proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and IMP-2 may be used tofacilitate purification. One such expression vector provides forexpression of a fusion protein containing IMP-2 and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinasecleavage site provides a means for purifying IMP-2 from the fusionprotein. A discussion of vectors which contain fusion proteins isprovided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

[0108] In addition to recombinant production, fragments of IMP-2 may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of IMP-2 may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0109] Therapeutics

[0110] Based on the chemical and structural homology among IMP-2 (SEQ IDNO: 1) and the mouse E25AMM protein (SEQ ID NO: 3), IMP-2 appears to bea member of the E25 multigene family. E25AMM has been shown to bedifferentially expressed in bone and cartilage tissue and thus serves asa marker for chondro-osteogenic differentiation (Deleersnijder W et al.,supra). Based on the homology between IMP-2 and E25AMM, IMP-2 maylikewise serve as a marker for bone and cartilage tissue. Moreimportantly, as shown herein, IMP-2 is strongly expressed in normaladult liver and its expression decreases precipitously in abnormal livertissues, including primary biliary cirrhosis and liver tumors. Thus,decreased or low level (i.e., less than about 50% the level seen innormal or disease-free, liver tissue) expression of IMP-2 in livertissue serves as an indicator of liver disease, including liver cancer.A similar decrease in IMP-2 transcripts is observed when normal lung andlung tumors are compared; lung tumors show about a 50% decrease in IMP-2transcript abundance as compared to normal adult lung. IMP-2 isexpressed in a variety of other tumor types including brain, prostate,breast and bladder.

[0111] Therefore, in one embodiment, IMP-2 or a fragment or derivativethereof may be administered to a subject to treat disorders associatedwith abnormal liver function as well as a variety of tumors. Suchconditions and diseases may include, but are not limited to, livertumors, primary biliary cirrohsis and lung, brain, prostate, breast andbladder tumors.

[0112] In another embodiment, a vector capable of expressing IMP-2, or afragment or a derivative thereof, may also be administered to a subjectto treat the liver tumors, primary biliary cirrohsis and lung, brain,prostate, breast and bladder tumors described above.

[0113] In another embodiment, IMP-2 may be administered in combinationwith other conventional chemotherapeutic agents. The combination oftherapeutic agents having different mechanisms of action will havesynergystic effects allowing for the use of lower effective doses ofeach agent and lessening side effects.

[0114] In one aspect, agonists of IMP-2 may be used to increase theactivity of IMP-2 in cells having reduced IMP-2 levels. Antibodies whichare specific for IMP-2 may be used directly as an agonist, or indirectlyas a targeting or delivery mechanism for bringing a pharmaceutical agentto cells or tissue which express IMP-2.

[0115] In one embodiment, antagonists or inhibitors of IMP-2 may beadministered to a subject to treat or prevent tumors, particularlybrain, prostate, breast and bladder tumors.

[0116] In another embodiment, a vector expressing antisense of thepolynucleotide encoding IMP-2 may be administered to a subject to treator prevent tumors, particularly brain, prostate, breast and bladdertumors.

[0117] Antagonists or inhibitors of IMP-2 may be produced using methodswhich are generally known in the art. In particular, purified IMP-2 maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind IMP-2.

[0118] Antibodies which are specific for IMP-2 may be used directly asan antagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express IMP-2.The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which reduce or abolish IMP-2 activity) are especially preferredfor therapeutic use.

[0119] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith IMP-2 or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0120] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to IMP-2 have an amino acid sequenceconsisting of at least five amino acids, and more preferably at least 10amino acids. It is also preferable that they are identical to a portionof the amino acid sequence of the natural protein, and they may containthe entire amino acid sequence of a small, naturally occurring molecule.Short stretches of IMP-2 amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0121] Monoclonal antibodies to IMP-2 may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0122] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceIMP-2-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0123] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0124] Antibody fragments which contain specific binding sites for IMP-2may also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0125] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between IMP-2 and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering IMP-2 epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0126] In another embodiment of the invention, the polynucleotidesencoding IMP-2, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding IMP-2 may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding IMP-2. Thus, antisense molecules may be used tomodulate IMP-2 activity, or to achieve regulation of gene function. Suchtechnology is now welt known in the art, and sense or antisenseoligomers or larger fragments, can be designed from various locationsalong the coding or control regions of sequences encoding IMP-2.

[0127] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingIMP-2. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

[0128] Genes encoding IMP-2 can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes IMP-2. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0129] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding IMP-2, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0130] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding IMP-2.

[0131] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0132] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding IMP-2. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0133] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0134] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0135] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0136] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of IMP-2,antibodies to IMP-2, mimetics, agonists, antagonists, or inhibitors ofIMP-2. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0137] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0138] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0139] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0140] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0141] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0142] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0143] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0144] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0145] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0146] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0147] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of IMP-2, such labeling wouldinclude amount, frequency, and method of administration.

[0148] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0149] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0150] A therapeutically effective dose refers to that amount of activeingredient, for example IMP-2 or fragments thereof, antibodies of IMP-2,agonists, antagonists or inhibitors of IMP-2, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.

[0151] Pharmaceutical compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0152] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0153] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0154] Diagnostics

[0155] In another embodiment, antibodies which specifically bind IMP-2may be used for the diagnosis of conditions or diseases characterized byexpression of IMP-2, or in assays to monitor patients being treated withIMP-2, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for IMP-2 includemethods which utilize the antibody and a label to detect IMP-2 in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0156] A variety of protocols including ELISA, RIA, and FACS formeasuring IMP-2 are known in the art and provide a basis for diagnosingaltered or abnormal levels of IMP-2 expression. Normal or standardvalues for IMP-2 expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to IMP-2 under conditions suitable for complex formation.The amount of standard complex formation may be quantified by variousmethods, but preferably by photometric means. Quantities of IMP-2expressed in subject, control, and disease samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0157] In another embodiment of the invention, the polynucleotidesencoding IMP-2 is used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofIMP-2 may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofIMP-2, and to monitor regulation of IMP-2 levels during therapeuticintervention.

[0158] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding IMP-2 or closely related molecules, may be used to identifynucleic acid sequences which encode IMP-2. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding IMP-2, alleles, or related sequences.

[0159] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the IMP-2 encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO: 2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring IMP-2.

[0160] Means for producing specific hybridization probes for DNAsencoding IMP-2 include the cloning of nucleic acid sequences encodingIMP-2 or IMP-2 derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as 32P or 35S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0161] Polynucleotide sequences encoding IMP-2 may be used for thediagnosis of conditions or diseases which are associated with expressionof IMP-2. Examples of such conditions or diseases include cancers of theliver, lung, brain, prostate, breast and bladder and primary biliarycirhorrsis. The polynucleotide sequences encoding IMP-2 may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered IMP-2 expression. Such qualitative or quantitative methods arewell known in the art.

[0162] In a particular aspect, the nucleotide sequences encoding IMP-2provide the basis for assays that detect activation or induction ofvarious cancers, particularly those mentioned above; in addition thelack of expression of IMP-2 may be detected using the IMP-2-encodingnucleotide sequences disclosed herein. The nucleotide sequences encodingIMP-2 may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding IMP-2 in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

[0163] In order to provide a basis for the diagnosis of diseaseassociated with expression of IMP-2, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes IMP-2,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0164] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0165] With respect to cancer, the presence of a relatively low or arelatively high amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

[0166] Additional diagnostic uses for oligonucleotides designed from thesequences encoding IMP-2 may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′->3′) and another withantisense (3′<-5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

[0167] Methods which may also be used to quantitate the expression ofIMP-2 include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P. C. et al.(1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.Biochem. 229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0168] In another embodiment of the invention, the nucleic acidsequences which encode IMP-2 may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0169] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding IMP-2 on a physical chromosomal map and a specific disease, or predisposition to a specific disease, may help delimit the regionof DNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0170] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0171] In another embodiment of the invention, IMP-2, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenIMP-2 and the agent being tested, may be measured.

[0172] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to IMP-2 largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with IMP-2, or fragments thereof, and washed.Bound IMP-2 is then detected by methods well known in the art. PurifiedIP-2 can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0173] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding IMP-2specifically compete with a test compound for binding IMP-2. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with IMP-2.

[0174] In additional embodiments, the nucleotide sequences which encodeIMP-2 may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0175] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0176] I Neutgmt01 cDNA Library Construction

[0177] The NEUTGMT01 library was constructed using 1 microgram of polyARNA isolated from peripheral blood granulocytes collected by densitygradient centrifugation through Ficoll-Hypaque. The cells were isolatedfrom buffy coat units obtained from 20 unrelated male and female donorsat the Stanford Blood Bank (Stanford, Calif.). Cells were cultured in 10nM GM-CSF for 1 hour before harvest.

[0178] Cells were lysed in buffer containing guanidinium isothiocyanate.The lysate was centrifuged over a 5.7 M CsCl cushion using a BeckmanSW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for18 hours at 25,000 rpm at ambient temperature. The RNA was extractedonce with acid phenol at pH 4.0, twice with phenol chloroform at pH 8.0,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in DEPC-treated water, and DNase treated for 15 min at 37°C. The RNA was isolated with the QIAGEN OLIGOTEX kit (QIAGEN Inc,Chatsworth Calif.) and used to construct the cDNA library.

[0179] The RNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Cat#18248-013; Gibco/BRL, Gaitherburg, Md.), and cDNAs were ligatedinto PSPORT1. The plasmid PSPORT1 was subsequently transformed intoDH5αD competent cells (Cat#18258-012, Gibco/BRL).

[0180] II Isolation and Sequencing of cDNA Clones

[0181] Plasmid DNA was released from the cells and purified using theMiniprep Kit (Cat# 77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96 well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Cat#22711, LIFE TECHNOLOGIES , GaithersburgMd.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteriawere cultured for 24 hours after the wells were inoculated and thenlysed with 60 μl of lysis buffer; 3) a centrifugation step employing theBeckman GS-6R at 2900 rpm for 5 min was performed before the contents ofthe block were added to the primary filter plate; and 4) the optionalstep of adding isopropanol to TRIS buffer was not routinely performed.After the last step in the protocol, samples were transferred to aBeckman 96-well block for storage.

[0182] Alternative methods of purifying plasmid DNA include the use ofMAGIC MINIPREPS-DNA, QIAwell—8 Plasmid, QIAwell PLUS DNA, and QIAwellULTRA DNA purification systems (QIAGEN Chatsworth Calif.).

[0183] The cDNAs were sequenced by the method of Sanger F and AR Coulson(1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton,Reno Nev.) in combination with four Peltier Thermal Cyclers (PTC200 fromMJ Research, Watertown Mass.) and Applied Biosystems 377 or 373 DNASequencing Systems (Perkin Elmer), and the reading frame was determined.

[0184] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0185] Each cDNA was compared to sequences in GenBank using a searchalgorithm developed by Applied Biosystems and incorporated into theINHERIT 670 sequence analysis system. In this algorithm, PatternSpecification Language (TRW Inc, Los Angeles, Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

[0186] Peptide and protein sequence homologies were ascertained usingthe INHERIT 670 sequence analysis system using the methods similar tothose used in DNA sequence homologies. Pattern Specification Languageand parameter windows were used to search protein databases forsequences containing regions of homology which were scored with aninitial value. Dot-matrix homology plots were examined to distinguishregions of significant homology from chance matches.

[0187] BLAST, which stands for Basic Local Alignment Search Tool(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990)J. Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

[0188] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0189] A comparison of the full-length and partial cDNA sequences andthe deduced amino acid sequences corresponding to the human IMP-2 geneand IMP-2 protein with known nucleotide and protein sequences in GenBankrevealed that the full-length human IMP-2 cDNA and protein sequences(i.e., SEQ ID NOS: 1 and 2) were unique (i.e., not previouslyidentified). This search revealed that the human IMP-2 protein sharedsome homology with the mouse E25AMM protein (SEQ ID NO: 3), theCaenorhabditis briggsae G01D9.4 gene product which is of unknownfunction (GI 1293791) and the HR21spa protein which is involved in DNAdouble-strand break repair (GI 1620398). In addition, portions of theamino acid sequence of IMP-2 were found to share homology with a numberof short EST sequences of human origin (GI 1331732, GI 1376262, GI764606, GI 873070 and GI 864758).

[0190] IV Northern Analysis

[0191] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0192] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ database (IncyteGenomics). This analysis is much faster than multiple, membrane-basedhybridizations. In addition, the sensitivity of the computer search canbe modified to determine whether any particular match is categorized asexact or homologous.

[0193] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0194] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0195] The results of northern analysis are reported as a list oflibraries in which the transcript encoding IMP-2 occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0196] Electronic northern analysis revealed that mRNA encoding humanIMP-2 (SEQ ID NO: 1) was present in libraries generated from a widevariety of adult and fetal tissues. IMP-2 cDNA is most stronglyexpressed in normal adult liver and its expression decreasesdramatically in abnormal liver tissues, including primary biliarycirrhosis and liver tumors. In addition to expression in apparentlynormal human tissues, IMP-2 was expressed in a variety of tumors,including but not limited to brain, prostate, breast and bladder tumorsas well as in several immortalized cell lines. IMP-2 cDNA is alsoexpressed in a variety of tissues and cell lines which are involved withthe immune response, including inflammatory and/or autoimmune disease(e.g., rheumatoid synovium, ulcerative colitis, Crohn's disease, primarybiliary cirrhosis).

[0197] V Extension of IMP-2-Encoding Polynucleotides to Full Length orto Recover Regulatory Sequences

[0198] Full length IMP-2-encoding nucleic acid sequence (SEQ ID NO: 2)is used to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0199] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0200] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M. J. Research,Watertown, Mass.) and the following; parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0201] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0202] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

[0203] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0204] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0205] VI Labeling and use of Hybridization Probes

[0206] Hybridization probes derived from SEQ ID NO: 2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ−³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified with Sephadex G-25superfine resin column (Pharmacia & Upjohn). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN).

[0207] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1 × salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0208] VII Antisense Molecules

[0209] Antisense molecules to the IMP-2-encoding sequence, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring IMP-2. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the coding sequences of IMP-2, as shown in FIGS. 1A and 1B is used toinhibit expression of naturally occurring IMP-2. The complementaryoligonucleotide is designed from the most unique 5′ sequence as shown inFIGS. 1A and 1B and used either to inhibit transcription by preventingpromoter binding to the upstream nontranslated sequence or translationof an IMP-2-encoding transcript by preventing the ribosome from binding.Using an appropriate portion of the signal and 5′ sequence of SEQ ID NO:2, an effective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the signal or 5′ codingsequence of the polypeptide as shown in FIGS. 1A and 1B.

[0210] VIII Expression of IMP-2

[0211] Expression of IMP-2 is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, PSPORT, previously used for thegeneration of the cDNA library, is used to express IMP-2 in E. coli.Upstream of the cloning site, this vector contains a promoter for6-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0212] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein or fragments thereof. Sequencesencoding IMP-2 fusion proteins lacking the hydrophobic stretches locatedat residues 53-76 of SEQ ID NO: 1 (this stretch may anchor IMP-2 in themembrane) are preferentially employed for the production of solubleforms of recombinant IMP-2. The signal residues present on the PSPORTvector direct the secretion of IMP-2 into the bacterial growth mediawhich can be used directly in the following assay for activity.

[0213] Alternatively, IMP-2 may be expressed as a membrane-bound proteinin a host cell and the recombinant IMP-2 recovered from the membrane ofthe host cell using techniques well known to the art .

[0214] IX Demonstration of IMP-2 Activity

[0215] Given the chemical and structural similarity between the humanIMP-2 and mouse E25AMM proteins, IMP-2 is presumed to be a type IIintegral membrane protein. To demonstrate that IMP-2 is an integralmembrane protein, sequences encoding IMP-2 are expressed in cells whichlack the ability to express IMP-2 and the location of IMP-2 isascertained using conventional techniques (e.g., immunoprecipitation ofproteins derived from cell membrane-containing fractions and solublefractions lacking membrane associated proteins; preparation ofanti-IMP-2 antibodies is described below). Expression of IMP-2 isachieved using methods known to the art as described above; numerousexpression vectors are available for the expression of proteins ineukaryotic and prokaryotic hosts. Cells which lack the ability toexpress human IP-2 are easily obtained as any non-human eukaryotic cellline is expected to lack the ability to express human IMP-2; inaddition, prokaryotic cells would lack the ability to express IMP-2.Confirmation that a cell lacks the ability to express IMP-2 is obtainedby a variety of means known to the art including Northern blot analysisin which RNA isolated from the candidate host cell is hybridized withIMP-2-encoding sequences (e.g., SEQ ID NO: 2); cells whose RNA fails tohybridize with IMP-2 sequences are suitable IMP-2-negative host cells.In addition, anti-IMP-2 antibodies can be used to confirm that thecandidate host cell lacks proteins which react or cross-react withIMP-2.

[0216] As described above, a reduction in IMP-2 expression is seen indiseased liver tissues. This suggests that increasing the expression ofIMP-2 in liver disease may have a therapeutic effect. Expression vectorscapable of directing the expression of IMP-2 in liver tissue (e.g.,using a liver-specific promoter such as the albumin promoter) are usedto increase the expression of IMP-2 in the liver of an animal sufferingfrom liver disease. A portion of the diseased liver is removed(explanted) and the expression vector is transferred into the explantedliver cells ex vivo and the cells containing the IMP-2 expression vectorare returned to the animal. Alternatively, IMP-2 encoding sequencesunder the control of a liver-specific promoter may be transferreddirectly to the liver of an animal using a suitable vector (e.g.,retroviral vectors, adenoviral vectors, direct injection of DNA, etc.)using techniques known to the art. Liver function is monitored inanimals receiving the IMP-2 sequences and in control animals which donot receive IMP-2 sequences (both diseased and disease-free or normalanimals are employed as controls); liver function may be assessed bymeasurement of the serum level of aminotransferases, bilirubin, albuminγ-globulin and measurement of the prothrombin time. An improvement inliver function in diseased animals which received IMP-2 sequencesindicates the therapeutic effect of increasing IMP-2 expression in liverdisease.

[0217] X Production of IMP-2 Specific Antibodies

[0218] IMP-2 that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO: 2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0219] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0220] XI Purification of Naturally Occurring IMP-2 using SpecificAntibodies

[0221] Naturally occurring or recombinant IMP-2 is substantiallypurified by immunoaffinity chromatography using antibodies specific forIMP-2. An immunoaffinity column is constructed by covalently couplingIMP-2 antibody to an activated chromatographic resin, such asCnBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, theresin is blocked and washed according to the manufacturer'sinstructions.

[0222] Media containing IMP-2 is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of IMP-2 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/IMP-2 binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and IMP-2 iscollected.

[0223] XII Identification of Molecules which Interact with IMP-2

[0224] MP-2 or biologically active fragments thereof are labeled with1251 Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled IMP-2, washed and any wells withlabeled IMP-2 complex are assayed. Data obtained using differentconcentrations of IMP-2 are used to calculate values for the number,affinity, and association of IMP-2 with the candidate molecules.

[0225] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 4 266 amino acids amino acid single linear NEUTGMT01 632664 1 Met ValLys Val Thr Phe Asn Ser Ala Leu Ala Gln Lys Glu Ala Lys 1 5 10 15 LysAsp Glu Pro Lys Ser Gly Glu Glu Ala Leu Ile Ile Pro Pro Asp 20 25 30 AlaVal Ala Val Asp Cys Lys Asp Pro Asp Asp Val Val Pro Val Gly 35 40 45 GlnArg Arg Ala Trp Cys Trp Cys Met Cys Phe Gly Leu Ala Phe Met 50 55 60 LeuAla Gly Val Ile Leu Gly Gly Ala Tyr Leu Tyr Lys Tyr Phe Ala 65 70 75 80Leu Gln Pro Asp Asp Val Tyr Tyr Cys Gly Ile Lys Tyr Ile Lys Asp 85 90 95Asp Val Ile Leu Asn Glu Pro Ser Ala Asp Ala Pro Ala Ala Leu Tyr 100 105110 Gln Thr Ile Glu Glu Asn Ile Lys Ile Phe Glu Glu Glu Glu Val Glu 115120 125 Phe Ile Ser Val Pro Val Pro Glu Phe Ala Asp Ser Asp Pro Ala Asn130 135 140 Ile Val His Asp Phe Asn Lys Lys Leu Thr Ala Tyr Leu Asp LeuAsn 145 150 155 160 Leu Asp Lys Cys Tyr Val Ile Pro Leu Asn Thr Ser IleVal Met Pro 165 170 175 Pro Arg Asn Leu Leu Glu Leu Leu Ile Asn Ile LysAla Gly Thr Tyr 180 185 190 Leu Pro Gln Ser Tyr Leu Ile His Glu His MetVal Ile Thr Asp Arg 195 200 205 Ile Glu Asn Ile Asp His Leu Gly Phe PheIle Tyr Arg Leu Cys His 210 215 220 Asp Lys Glu Thr Tyr Lys Leu Gln ArgArg Glu Thr Ile Lys Gly Ile 225 230 235 240 Gln Lys Arg Glu Ala Ser AsnCys Phe Ala Ile Arg His Phe Glu Asn 245 250 255 Lys Phe Ala Val Glu ThrLeu Ile Cys Ser 260 265 1114 base pairs nucleic acid single linearNEUTGMT01 632664 2 GCCGCCTCTG CCGCCGCGGA CTTCCCGAAC CTCTTCAGCCGCCCGGAGCC GCTCCCGGAG 60 CCCGGCCGTA GAGGCTGCAA TCGCAGCCGG TGAGCCCGCAGCCCGCGCCC CGAGCCCGCC 120 GCCGCCCTTC GAGGGCGCCC CAGGCCGCGC CATGGTGAAGGTGACGTTCA ACTCCGCTCT 180 GGCCCAGAAG GAGGCCAAGA AGGACGAGCC CAAGAGCGGCGAGGAGGCGC TCATCATCCC 240 CCCCGACGCC GTCGCGGTGG ACTGCAAGGA CCCAGATGATGTGGTACCAG TTGGCCAAAG 300 AAGAGCCTGG TGTTGGTGCA TGTGCTTTGG ACTAGCATTTATGCTTGCAG GTGTTATTCT 360 AGGAGGAGCA TACTTGTACA AATATTTTGC ACTTCAACCAGATGACGTGT ACTACTGTGG 420 AATAAAGTAC ATCAAAGATG ATGTCATCTT AAATGAGCCCTCTGCAGATG CCCCAGCTGC 480 TCTCTACCAG ACAATTGAAG AAAATATTAA AATCTTTGAAGAAGAAGAAG TTGAATTTAT 540 CAGTGTGCCT GTCCCAGAGT TTGCAGATAG TGATCCTGCCAACATTGTTC ATGACTTTAA 600 CAAGAAACTT ACAGCCTATT TAGATCTTAA CCTGGATAAGTGCTATGTGA TCCCTCTGAA 660 CACTTCCATT GTTATGCCAC CCAGAAACCT ACTGGAGTTACTTATTAACA TCAAGGCTGG 720 AACCTATTTG CCTCAGTCCT ATCTGATTCA TGAGCACATGGTTATTACTG ATCGCATTGA 780 AAACATTGAT CACCTGGGTT TCTTTATTTA TCGACTGTGTCATGACAAGG AAACTTACAA 840 ACTGCAACGC AGAGAAACTA TTAAAGGTAT TCAGAAACGTGAAGCCAGCA ATTGTTTCGC 900 AATTCGGCAT TTTGAAAACA AATTTGCCGT GGAAACTTTAATTTGTTCTT GAACAGTCAA 960 GAAAAACATT ATTGAGGAAA ATTAATATCA CAGCATAACCCCACCCTTTA CATTTTGTGC 1020 AGTGATTATT TTTTAAAGTC TTCTTTCATG TAAGTAGCAAACAGGGCTTT ACTATCTTTT 1080 CATCTCATTA ATTCAATTAA AACCATTACC TTAA 1114263 amino acids amino acid single linear GenBank 624778 3 Met Val LysIle Ala Phe Asn Thr Pro Thr Ala Val Gln Lys Glu Glu 1 5 10 15 Ala ArgGln Asp Ile Glu Ala Leu Val Ser Arg Thr Val Arg Ala Gln 20 25 30 Ile LeuThr Gly Lys Glu Leu Arg Val Val Pro Gln Glu Lys Asp Gly 35 40 45 Ser SerGly Arg Cys Met Leu Thr Leu Leu Gly Leu Ser Phe Ile Leu 50 55 60 Ala GlyLeu Ile Val Gly Gly Ala Cys Ile Tyr Lys Tyr Phe Met Pro 65 70 75 80 LysSer Thr Ile Tyr His Gly Glu Met Cys Phe Phe Asp Ser Glu Asp 85 90 95 ProVal Asn Ser Ile Pro Gly Gly Glu Pro Tyr Phe Leu Pro Val Thr 100 105 110Glu Glu Ala Asp Ile Arg Glu Asp Asp Asn Ile Ala Ile Ile Asp Val 115 120125 Pro Val Pro Ser Phe Ser Asp Ser Asp Pro Ala Ala Ile Ile His Asp 130135 140 Phe Glu Lys Gly Met Thr Ala Tyr Leu Asp Leu Leu Leu Gly Asn Cys145 150 155 160 Tyr Leu Met Pro Leu Asn Thr Ser Ile Val Met Thr Pro LysAsn Leu 165 170 175 Val Glu Leu Phe Gly Lys Leu Ala Ser Gly Lys Tyr LeuPro His Thr 180 185 190 Tyr Val Val Arg Glu Asp Leu Val Ala Val Glu GluIle Arg Asp Val 195 200 205 Ser Asn Leu Gly Ile Phe Ile Tyr Gln Leu CysAsn Asn Arg Lys Ser 210 215 220 Phe Arg Leu Arg Arg Arg Asp Leu Leu LeuGly Phe Asn Lys Arg Ala 225 230 235 240 Ile Asp Lys Cys Trp Lys Ile ArgHis Phe Pro Asn Glu Phe Ile Val 245 250 255 Glu Thr Lys Ile Cys Gln Glu260 1635 base pairs nucleic acid single linear GenBank 624777 4GGGAGACCTG AGCTCGCTGC TGCCTGTGGA AGACTGGGAG AGGAGACACT AAGTGCTGCT 60CAAGCAAGCG CGATCCTCTC CTCTTTCAAC CTGCAGCCCA AGATACTGAT TCGAGCCGCG 120CCTTACCGCG CAGCCCGAAG ATTCACCATG GTGAAGATCG CCTTCAACAC CCCTACGGCG 180GTGCAAAAGG AGGAGGCGCG GCAAGATATA GAGGCGCTCG TCAGTCGCAC TGTCCGAGCT 240CAAATCCTGA CTGGCAAGGA GCTCAGAGTT GTCCCGCAGG AGAAAGATGG CTCATCTGGG 300AGATGCATGC TTACTCTCCT AGGCCTCTCA TTCATCTTGG CAGGACTGAT TGTTGGTGGA 360GCCTGCATTT ACAAGTACTT CATGCCCAAG AGCACCATTT ACCATGGTGA GATGTGCTTC 420TTTGATTCTG AGGATCCTGT CAATTCCATT CCTGGAGGAG AGCCATACTT TCTGCCTGTG 480ACTGAGGAGG CTGATATCCG TGAGGATGAC AACATTGCCA TCATTGATGT GCCTGTGCCC 540AGTTTCTCTG ATAGCGATCC GGCGGCAATT ATTCACGACT TTGAGAAGGG AATGACTGCT 600TACCTGGACT TGCTTTTGGG AAACTGTTAT CTGATGCCCC TCAATACTTC CATTGTTATG 660ACTCCAAAGA ATCTGGTGGA ACTTTTTGGA AAACTGGCAA GTGGCAAGTA TTTGCCTCAT 720ACTTATGTGG TTCGTGAAGA CCTGGTTGCT GTGGAAGAAA TTCGTGATGT TAGTAACCTT 780GGTATTTTTA TTTACCAACT TTGCAACAAC CGAAAATCCT TCCGCCTTAG ACGCAGAGAC 840CTTCTGCTGG GTTTCAACAA GCGTGCCATT GACAAATGCT GGAAGATTAG ACACTTCCCC 900AATGAATTTA TCGTTGAAAC CAAGATCTGT CAGGAGTGAA ATGTGACAGA TAAAGAGTAT 960CCTTGATAAT AAGAAGTCAG GAACTTACCG TCTGACTTGG AAAATTGAAA TTGATGGGAT 1020ACTCATGCTA TTTACTCATA CATTTACTCT ATTGCTTATA CTGGAAAAGG AAAGGGAAAG 1080GGGGGAGAAA ACTACTAACC ACTGCAAGCG ATTGTCCAAT TCTACTTTAA TTGACATTGC 1140TTGCTGTTTT CAACAAGTCA AATGATTATC TTTTCTCTTG AATTTATAGG GTTTAGATTT 1200CTGAAAGCAG CATGAATGTG TCATCTTACC ATCCTGACAA TAAAGCCCAT CCTCTGGTTT 1260TATTTAAAGC AAGCTCTTTC CAACATCACT TGGCTAGAGC ATGCTTTAAA TTTAAAATAT 1320TTGAAATTTG TTTTTGACAT TTTTTTGTGT GAAACATGTC AAATCTCTTA CCATTCTTTG 1380GTTTTCTTCT TTATTATGTT CAACTCTCCT GATTTCAGAA GTTACATTTT TGCATTTCTA 1440TCAGGTGCTG TGTAACGAAT CTGACTGATA TGTGAACAAT CTTCATGAGG AAGCAATTTT 1500TTACTCATGT AATGATTCTT TCTCACTGAT ATCTGTATTG TGAAATCCAC AGAACTGTAC 1560AGGTGCTGAA TGCTGTAAGG AGTTCTGGTT GTATGAATTC TACAACCCTA TAATAAAGTT 1620TACCGTATTC AATCA 1635

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO: 1, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, c) a biologically active fragment of a polypeptide having an aminoacid sequence of SEQ ID NO: 1, and d) an immunogenic fragment of apolypeptide having an amino acid sequence of SEQ ID NO:
 1. 2. Anisolated polypeptide of claim 1, comprising the amino acid sequence ofSEQ ID NO:
 1. 3. An isolated polynucleotide encoding a polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim2.
 5. An isolated polynucleotide of claim 4, having a sequence of SEQ IDNO:
 2. 6. A recombinant polynucleotide comprising a promoter sequenceoperably linked to a polynucleotide of claim
 3. 7. A cell transformedwith a recombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method ofproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:
 1. 11. An isolated antibody which specifically binds to apolypeptide of claim
 1. 12. An isolated polynucleotide selected from thegroup consisting of: a) a polynucleotide comprising a polynucleotidesequence of SEQ ID NO: 2, b) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 90% identical to apolynucleotide sequence of SEQ ID NO: 2, c) a polynucleotidecomplementary to a polynucleotide of a), d) a polynucleotidecomplementary to a polynucleotide of b) and e) an RNA equivalent ofa)-d).
 13. An isolated polynucleotide comprising at least 60 contiguousnucleotides of a polynucleotide of claim
 12. 14. A method of detecting atarget polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide of claim 12, the method comprising: a)hybridizing the sample with a probe comprising at least 20 contiguousnucleotides comprising a sequence complementary to said targetpolynucleotide in the sample, and which probe specifically hybridizes tosaid target polynucleotide, under conditions whereby a hybridizationcomplex is formed between said probe and said target polynucleotide orfragments thereof, and b) detecting the presence or absence of saidhybridization complex, and, optionally, if present, the amount thereof.15. A method of claim 14, wherein the probe comprises at least 60contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence of SEQ ID NO:
 1. 19. A method for treating a disease orcondition associated with decreased expression of functional IMP-2,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional IMP-2, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional IMP-2, comprising administering to apatient in need of such treatment a composition of claim
 24. 26. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, saidmethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method of assessing toxicityof a test compound, the method comprising: a) treating a biologicalsample containing nucleic acids with the test compound, b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of IMP-2 in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of IMP-2 in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofIMP-2 in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of an amino acid sequence of SEQ ID NO: 1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibodies from said animal, and c) screening theisolated antibodies with the polypeptide, thereby identifying apolyclonal antibody which binds specifically to a polypeptide comprisingan amino acid sequence of SEQ ID NO:
 1. 37. A polyclonal antibodyproduced by a method of claim
 36. 38. A composition comprising thepolyclonal antibody of claim 37 and a suitable carrier.
 39. A method ofmaking a monoclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of an amino acid sequence of SEQ ID NO: 1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibody producing cells from the animal, c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells, d) culturing thehybridoma cells, and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide comprising an amino acidsequence of SEQ ID NO:
 1. 40. A monoclonal antibody produced by a methodof claim
 39. 41. A composition comprising the monoclonal antibody ofclaim 40 and a suitable carrier.
 42. The antibody of claim 11, whereinthe monoclonal antibody is produced by screening a Fab expressionlibrary.
 43. The antibody of claim 11, wherein the antibody is producedby screening a recombinant immunoglobulin library.
 44. A method ofdetecting a polypeptide comprising an amino acid sequence of SEQ ID NO:1 in a sample, the method comprising: a) incubating the antibody ofclaim 11 with a sample under conditions to allow specific binding of theantibody and the polypeptide, and b) detecting specific binding, whereinspecific binding indicates the presence of a polypeptide comprising anamino acid sequence of SEQ ID NO: I in the sample.
 45. A method ofpurifying a polypeptide comprising an amino acid sequence of SEQ ID NO:1 from a sample, the method comprising: a) incubating the antibody ofclaim 11 with a sample under conditions to allow specific binding of theantibody and the polypeptide, and b) separating the antibody from thesample and obtaining the purified polypeptide comprising an amino acidsequence of SEQ ID NO:
 1. 46. A microarray wherein at least one elementof the microarray is a polynucleotide of claim
 13. 47. A method ofgenerating an expression profile of a sample which containspolynucleotides, the method comprising: a) labeling the polynucleotidesof the sample, b) contacting the elements of the microarray of claim 46with the labeled polynucleotides of the sample under conditions suitablefor the formation of a hybridization complex, and c) quantifying theexpression of the polynucleotides in the sample.
 48. An array comprisingdifferent nucleotide molecules affixed in distinct physical locations ona solid substrate, wherein at least one of said nucleotide moleculescomprises a first oligonucleotide or polynucleotide sequencespecifically hybridizable with at least 30 contiguous nucleotides of atarget polynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:
 1. 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO: 2.